The present invention relates to improved and optimized methods for direct labeling of proteins, especially antibodies and/or antibody fragments, with radioisotopes of technetium and rhenium.
The isotope Technetium-99m is among the most valuable in diagnostic nuclear medicine due to its ready availability, low cost and favorable radiochemical characteristics. It is used widely as an agent for labeling macromolecules such as monoclonal antibodies and can be bound to the protein in various ways. Early work mainly used the bifunctional chelate approach, i.e., use of a chelator which contained another functional group for linkage to the protein. Various forms of diethylenetriaminepentaacetic acid (DTPA) were used, for example, to bind to the antibody and also to chelate the radiometal ion.
Direct labeling of protein was also tried, using a "pretinning" protocol, requiring severe conditions and long "pretinning" times, but radiolabeling at 100% incorporation was not achieved. Moreover, the presence of extremely high amounts of stannous ion for long periods compromised the immunoreactivity of the antibody. The process also generally necessitated a post-labeling purification column. Attempts to repeat pretinning procedures of others with F(ab').sub.2 antibody fragments were unsatisfactory in achieving Tc-99m labeling.
Other, more recent direct labeling methods have required separate vials, one for antibody and one for stannous ion complexed to a transchelator such as a phosphate and/or phosphonate.
The element below technetium in the periodic table, rhenium, has similar chemical properties and might be expected to react in an analogous manner to technetium. There are some 34 isotopes of rhenium and two of them in particular, rhenium-186 (t 1/2, 90h; gamma 137 keV, beta 1.07, 0.93 MeV) and rhenium-188 (t 1/2, 17h; gamma 155 keV, beta 2.12 MeV), are prime candidates for radioimmunotherapy using monoclonal antibody approaches. Both isotopes also have gamma emissions at suitable energies for gamma camera imaging purposes. Rhenium-186 is obtained from reactor facilities by bombardment of enriched rhenium-185 with neutrons, which yields rhenium-186 in a "carrier-added" form containing a large excess of non-radioactive rhenium-185. Rhenium-188 is obtained from a tungsten-188/rhenium-188 generator (Oak Ridge National Laboratory) and can be eluted from the generator in a substantially carrier-free form with little tungsten breakthrough. Also, the energy deposition from this isotope at a high .DELTA.=1.63 g-rad/.mu.Ci-h is close to another potently energetic potential therapeutic, yttrium- 90 (.DELTA.=1.99 g-rad/.mu.Ci-h) while at the same time the chemical properties of rhenium may make it less of a bone-seeking agent than yttrium (which is often contaminated with strontium-90) and give rise to better tumor/organ biodistribution and dosimetry.
Although many groups have alluded to the possibility of utilizing rhenium to label antibodies in the same fashion as technetium, little successful work has been published. Low rhenium incorporations are usually seen with antibody-chelate conjugates and there is a general tendency of rhenium to reoxidize back to perrhenate and then dissociate from complexation. Besides, use of the bifunctional chelate approach often requires an organic synthesis with a lengthy series of intermediates to be isolated and purified prior to antibody conjugation.
Numerous attempts by the inventor and others to produce a F(ab)'.sub.2 fragment radiolabeled with technetium or rhenium, and substantially free of Fab' fragments were unsuccessful. Such a conjugate is desirable for its advantageous combination of clearance rate, extent of tumor localisation and biodistribution.
A need continues to exist for simple, efficient methods for radiolabeling proteins with radioisotopes of technetium and rhenium.